Registration Dossier

Diss Factsheets

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 Oct 2014 - 02 Feb 2015
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
according to guideline
other: Commission Regulation (EU) No 640/2012 of 6 July 2012 amending, for the purpose of its adaptation to technical progress, Regulation (EC) Nº 440/2008, B.46 B.46: In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Physical state: Solid / yellow
- Storage condition of test material: Room temperature

In vitro test system

Test system:
human skin model
Three dimensional human epidermis model EpiDermTM
Source species:
Cell type:
non-transformed keratinocytes
Details on test system:
The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Amount/concentration applied:
25 μL sterile PBS was applied first. Thereafter, a bulk volume of 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed together with the fluid. A nylon mesh was placed carefully onto the tissue surface afterwards.
Duration of treatment / exposure:
The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator (1h exposure total).
Number of replicates:
Three tissues were treated with the test substance, the PC and NC, respectively.

Test system

other: Control tissues were concurrently treated with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC).
Details on study design:
The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were placed into the incubator at 37°C for 24 ± 2 hours.
After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period. After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Results and discussion

In vitro

Irritation / corrosion parameter:
% tissue viability
mean of 3 tissues
Run / experiment:
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The test substance is not able to reduce MTT directly, as determined in a pretest.

Any other information on results incl. tables

Individual and mean OD570 values, individual and mean viability values and standard deviations

substance   tissue 1 tissue 2 tissue 3 mean SD
NC mean OD570 2.809 2.714 2.843 2.789 0.067
viability [% of NC] 100.7 97.3 101.9 100 2.4
test substance mean OD570 2.931 2.561 2.979 2.824 0.229
viability [% of NC] 105.1 91.8 106.8 101 8.2
PC mean OD570 0.101 0.089 0.115 0.101 0.013
viability [% of NC] 3.6 3.2 4.1 4 0.5

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
The test article does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.
Executive summary:

The potential of the test article to cause dermal corrosion/irritation was assessed by a single topical application of 25 μL bulk volume (about 14 mg) of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). The irritation test was performed with three EpiDerm™ tissue samples, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The EpiDerm™ skin irritation test showed the following results: The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 101%. Based on the observed results it was concluded, that the test article does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Categories Display